PROJECT SUMMARY/ABSTRACT Heterochromatin plays critical roles in maintaining genome stability and in regulating transcriptional gene silencing (TGS) during development. It has become increasingly clear that misregulation of pathways influencing heterochromatin integrity cause or contribute to many human maladies, including numerous cancers. Heterochromatin establishment, TGS, and epigenetic inheritance are complex processes regulated by numerous chromatin-associated factors. While core principles of heterochromatin biology have been suggested, most remain speculative. The proposed work will experimentally test and articulate these core principles by distilling essential features of heterochromatin in a highly-controlled and orthogonal environment. This research plan is composed of four aims and expands on the successful reconstitution of human-like histone 3 lysine 9 methylation (H3K9me)-dependent heterochromatin in Saccharomyces cerevisiae cells, which naturally lack H3K9me. Aim 1 is to determine the structural basis for H3K9me deposition by utilizing in vivo photo-cross-linking strategies and single particle cryo-electron microscopy. Aim 2 is to reconstitute high- fidelity epigenetic inheritance by performing high-throughput genetic screens to comprehensively identify S. cerevisiae modulators of H3K9me-dependent heterochromatin maintenance and by investigating the effect of heterochromatin domain size on the heritability of silent chromatin states. Aim 3 is to reconstitute human-like histone 3 lysine 27 methylation (H3K27me)-dependent heterochromatin in S. cerevisiae cells, which also naturally lack H3K27me, with the goal of defining the minimal requirements for establishing a repressive chromatin state that is crucial for the silencing of developmentally regulated genes in metazoans. Aim 4 is to investigate heterochromatin-dependent epigenetic adaptation by providing S. cerevisiae cells with heterologous gene silencing systems and determining how cells appropriate these systems to adapt to environmental stress in a DNA sequence-independent manner. The research plan will combine synthetic biology, mass-spectrometry, structural biology, and next generation sequencing experimental approaches to transform our mechanistic understanding of heterochromatin formation and to establish principles of epigenetic adaption. The proposed work will thus pave the way for discoveries that have meaningful implications for the study of evolution and development while opening new avenues to the treatment of human disease. In addition to scientific aims, I have also proposed a comprehensive training program (K99-phase) that will prepare me for research as an independent investigator (R00-phase). This program incorporates guidance from an advisory committee composed of renowned mentors and collaborators, acquisition of new skills related to single particle cryo-electron microscopy and fluorescence-activated cell sorting, training in the operation of state-of-the-art equipment at Harvard Medical School, as well as development of professional skills that will collectively facilitate my transition to independence.